This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Proteolytic processing is a key post-translational modification in blood plasma and serum. Most secreted proteins are processed by proteases at least once, when the signal peptide is removed, and additional processing occurs in many cases, such as prohormone activation. Also, intracellular proteins released into the blood may be processed by proteases, and this processing may be indicative of physiological status of cells in the body;for example, caspase-cleaved proteins in the blood may serve as markers of apoptosis. We have developed a method to profile proteolytic processing in human serum or plasma, based on specific labeling and enrichment of N-termini using an engineered enzyme called subtiligase. After enrichment, N-terminal peptides from proteolytically processed proteins can be identified by LCMSMS. This identification is absolutely depended on sensitive and high-resolution mass spectrometers including the Qstar Elite and Orbitrap instruments in the UCSF Mass Spectrometry Facility. We have identified nearly 1000 unique N-termini in about 300 proteins in whole serum and/or plasma. The identified proteins span nine orders of magnitude in plasma abundance, and include processing sites in complement and coagulation factors, annotated signal sequences, and processing sites in hormones and growth factors, including Gastric Inhibitory Peptide and Vascular Endothelial Growth Factor. Specific proteolytically cleaved peptides are good biomarker candidates and we are currently conducting a pilot study to discover peptide biomarkers of treatment efficacy in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL).